Botulinum neurotoxins (BoNTs) are a family of bacterial toxins that block synaptic vesicle exocytosis. Two types of BoNTs (BoNT/A and B) are now widely used to treat a growing list of medical conditions. As the use of BoNTs grows, major limitations and adverse effects have been identified including (1) diffusion of injected toxins to other regions, which is the cause of frequent occurrences of a wide range of non-life-threatening adverse effects, as well as serious consequences including death in rare occasions; (2) generation of neutralizing antibodies in patients that renders future treatment ineffective. Both issues are directly related to injection doses. Accordingly, enhancing the efficacy and specificity of BoNTs would decrease the required toxin doses in treatment and reduce the occurrence of adverse events in millions of patients. Indeed, the binding of BoNTs to human neurons can be improved, as we recently discovered that BoNT/B cannot bind to the major human receptor synaptotagmin II (Syt II) due to a single residue change in human Syt II sequence. This human receptor defect raises the need and also presents an opportunity for engineering the BoNT/B receptor binding domain to restore its binding to human Syt II. Such a modified BoNT/B receptor binding domain will significantly improve the efficacy of BoNT/B in humans and reduce toxin diffusion/immune response. Here we propose to carry out rational design mutagenesis in the BoNT/B receptor binding domain, using the co-crystal structure of BoNT/B-Syt II complexes as a guide, to identify specific mutations that restore BoNT/B binding to human Syt II. Once such mutations are identified, we will further characterize their binding to human Syt II in test tubes and on neuronal surfaces. These studies will generate modified BoNT/B receptor binding domains with significantly improved efficacy for binding human neurons, which will directly lead to the creation of a new generation of therapeutic toxins with improved efficacy, specificity, and safety.